3-Deoxyglucosone is a Potent Protein Glycating Agent Associated with Protein Crosslinking
3-deoxyglucosone (3DG) is a 1,2-dicarbonyl-3-deoxysugar which is a potent protein crosslinker, is teratogenic and/or mutagenic, causes apoptosis, mutations, and formation of active oxygen species, and is a precursor to the formation of Advanced Glycation End product (AGE) modified proteins. As reviewed by Brownlee and shown in FIG. 1, the previously generally accepted pathway for formation of 3DG comprises a reversible reaction between glucose and the ε-NH2 groups of lysine-containing proteins, forming a Schiff base (Brownlee et al., 1994, Diabetes 43:836-841). This Schiff base then rearranges to form a more stable ketoamine known as fructoselysine (FL) or the “Amadori product”. The dogma has been that 3DG production resulted exclusively from subsequent non-enzymatic rearrangement, dehydration, and fragmentation of the fructoselysine containing protein (Brownlee et al., 1994, Diabetes 43:836-841 and Makita et al., 1992, Science 258:651-653) (see FIG. 1). However, more recent work has shown that an enzymatic pathway for the production of 3DG exists as well (see FIGS. 1 and 2 and Brown et al., U.S. Pat. No. 6,004,958).
A metabolic pathway was discovered which produces relatively high concentrations of 3DG in organs affected by diabetes (Brown et al., U.S. Pat. No. 6,004,958). It was found that a specific kinase converts fructose-lysine into fructose-lysine-3-phosphate (FL3P) in an ATP dependent reaction, and that FL3P then breaks down to form free lysine, inorganic phosphate, and 3DG (Brown et al., U.S. Pat. No. 6,004,958). Methods have also been described for assessing diabetic risk, based on measuring components of the 3DG pathway (WO 99/64561).
U.S. Pat. No. 6,004,958 describes a class of compounds which inhibit the enzymatic conversion of fructose-lysine to FL3P and inhibit formation of 3DG. Specific compounds which are representative of the class have also been described (Brown et al., WO 98/33492). For example, it was found in WO 98/33492 that urinary or plasma 3DG can be reduced by meglumine, sorbitollysine, mannitollysine, and galactitollysine. It was also found in WO 98/33492 that diets high in glycated protein are harmful to the kidney and cause a decrease in birth rate.
It has also been disclosed that the fructoselysine pathway is involved in kidney carcinogenesis (WO 98/33492) and that diet and 3DG may play a role in carcinogenesis associated with this pathway (WO 00/24405; WO 00/62626).
Human skin is a composite material comprising a superficial component, the epidermis, and a deep component, the dermis. The outermost layer of the epidermis is the stratum corneum. This layer is the stiffest layer of the skin, as well as the one most affected by the surrounding environment. Deep to the stratum corneum is the internal portion of the epidermis. Deep to the epidermis, is the papillary layer of the dermis, which comprises relatively loose connective tissue which defines the micro-relief of the skin. The reticular dermis, deep to the papillary dermis, is dense connective tissue that is spatially organized. The reticular dermis is also associated with coarse wrinkles. Deep to the dermis is subcutaneous connective tissue and adipose tissue.
The principal functions of the skin include protection, excretion, secretion, absorption, thermoregulation, pigmentogenesis, accumulation, sensory perception, and regulation of immunological processes. These functions are detrimentally affected by the structural changes in the skin due to aging and various diseases and disorders of the skin. The physiological changes associated with normal skin aging and photoaging include loss of elasticity, decreased collagen, collagen and elastin crosslinking, wrinkling, dry/rough texture, and mottled hyperpigmentation, for example.
The mechanical properties of the skin, such as elasticity, are controlled by the density of the network of collagen and elastic fibers coursing throughout. Damaged collagen and elastin proteins lose their contractile properties, resulting in such things as skin wrinkling and skin surface roughness. As skin ages or begins to deteriorate due to a disease or disorder, it acquires sags, stretch marks, bumps, or wrinkles, it roughens, it can become discolored, and it has reduced ability to synthesize vitamin D. Aged skin also becomes thinner and has a flattened dermoepidermal interface because of the alterations of collagen, elastin, and glycosaminoglycans.
Experimental and Clinical Evidence of the Role of 3DG in Diabetes and Other Diseases
Past studies have concentrated on the role of 3DG in diabetes. It has been shown that diabetic humans have elevated levels of 3DG and 3-deoxyfructose (3DF), 3DG's detoxification product, in plasma (Niwa et al., 1993, Biochem. Biophys. Res. Commun. 196:837-843; Wells-Knecht et al., 1994, Diabetes. 43:1152-1156) and in urine (Wells-Knecht et al., 1994, Diabetes. 43:1152-1156), as compared with non-diabetic individuals. Furthermore, diabetics with nephropathy were found to have elevated plasma levels of 3DG compared to non-diabetics (Niwa et al., 1993, Biochem. Biophys. Res. Commun. 196:837-843). A recent study comparing patients with insulin-dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM) confirmed that 3DG and 3DF levels were elevated in blood and urine from both types of patient populations (Lal et al., 1995, Arch. Biochem. Biophys. 318:191-199). It has even been shown that incubation of glucose and proteins in vitro under physiological conditions produces 3DG. In turn, it has been demonstrated that 3DG glycates and crosslinks protein creating detectable AGE products (Baynes et al., 1984, Methods Enzymol. 106:88-98; Dyer et al., 1991, J. Biol. Chem. 266:11654-11660). The normal pathway for reductive detoxification of 3DG (conversion to 3DF) may be impaired in diabetic humans since their ratio of urinary and plasma 3DG to 3DF differs significantly from non-diabetic individuals (Lal et al., 1995, Arch Biochem. Biophys. 318:191-199).
Furthermore, elevated levels of 3DG-modified proteins have been found in diabetic rat kidneys compared to control rat kidneys (Niwa et al., 1997, J. Clin. Invest. 99:1272-1280). It has been demonstrated that 3DG has the ability to inactivate enzymes such as glutathione reductase, a central antioxidant enzyme. It has also been shown that Hemoglobin-AGE levels are elevated in diabetic individuals (Makita et al., 1992, Science 258:651-653) and other AGE proteins have been shown in experimental models to accumulate with time, increasing from 5-50 fold over periods of 5-20 weeks in the retina, lens and renal cortex of diabetic rats (Brownlee et al., 1994, Diabetes 43:836-841). In addition, it has been demonstrated that 3DG is a teratogenic factor in diabetic embryopathy (Eriksson et al., 1998, Diabetes 47:1960-1966).
Nonenzymatic glycation, in which reducing sugars are covalently attached to free amino groups and ultimately form AGEs, has been found to occur during normal aging and to occur at an accelerated rate in diabetes mellitus (Bierhaus et al., 1998, Cardiovasc. Res. 37:586-600). Crosslinking of proteins and the subsequent AGE formation are irreversible processes that alter the structural and functional properties of proteins, lipid components, and nucleic acids (Bierhaus et al., 1998, Cardiovasc. Res. 37:586-600). These processes have been postulated to contribute to the development of a range of diabetic complications including nephropathy, retinopathy, and neuropathy (Rahbar et al., 1999, Biochem. Biophys. Res. Commun. 262:651-660).
Recently, it has been demonstrated that inhibition of AGE formation reduced the extent of nephropathy in diabetic rats (Ninomiya et al., 2001, Diabetes 50:178-179). Therefore, substances which inhibit AGE formation and/or oxidative stress appear to limit the progression of diabetes and its complications and may offer new tools for therapeutic interventions in the therapy of diabetes (Bierhaus et al., 1998, Cardiovasc. Res. 37:586-600; Thornalley, 1996, Endocrinol. Metab. 3:149-166).
Detoxification of 3DG
3DG can be detoxified in the body by at least two pathways. In one pathway, 3DG is reduced to 3-deoxyfructose (3DF) by aldehyde reductase, and the 3DF is then efficiently excreted in urine (Takahashi et al., 1995, Biochemistry 34:1433). Another detoxification reaction oxidizes 3DG to 3-deoxy-2-ketogluconic acid (DGA) by oxoaldehyde dehydrogenase (Fujii et al., 1995, Biochem. Biophys. Res. Comm. 210:852).
Results of studies to date show that the efficiency of at least one of these enzymes, aldehyde reductase, is adversely affected in diabetes. When isolated from diabetic rat liver, this enzyme is glycated on lysine at positions 67, 84 and 140 and has a low catalytic efficiency when compared with the normal, unmodified enzyme (Takahashi et al., 1995, Biochemistry 34:1433). Since diabetic patients have higher ratios of glycated proteins than normoglycemic individuals they are likely to have both higher levels of 3DG and a reduced ability to detoxify this reactive molecule by reduction to 3DF. It has also been found that overexpression of aldehyde reductase protects PC12 cells from the cytotoxic effects of methylglyoxal or 3DG (Suzuki et al., 1998, J. Biochem. 123:353-357).
The mechanism by which aldehyde reductase works has been studied. These studies demonstrated that this important detoxification enzyme is inhibited by aldose reductase inhibitors (ARIs) (Barski et al., 1995, Biochemistry 34:11264). ARIs are currently under clinical investigation for their potential to reduce diabetic complications. These compounds, as a class, have shown some effect on short term diabetic complications. However, they lack clinical effect on long term diabetic complications and they worsen kidney function in rats fed a high protein diet. This finding is consistent with the newly discovered metabolic pathway for lysine recovery. For example, a high protein diet will increase the consumption of fructose-lysine, which in turn undergoes conversion into 3DG by the kidney lysine recovery pathway. The detoxification of the resulting 3DG by reduction to 3DF will be inhibited by ARIs therapy. Inhibiting 3DG detoxification will lead to increased 3DG levels, with a concomitant increase in kidney damage, as compared to rats not receiving AR's. This is because inhibition of the aldose reductase by the AR's would reduce availability of aldose reductase for reducing 3DG and 3DF.
Aminoguanidine, an agent which detoxifies 3DG pharmacologically via formation of rapidly excreted covalent derivatives (Hirsch et al., 1992, Carbohydr. Res. 232:125-130), has been shown to reduce AGE-associated retinal, neural, arterial, and renal pathologies in animal models (Brownlee et al., 1994, Diabetes 43:836-841; Brownlee et al., 1986, Science 232:1629-1632; Ellis et al., 1991, Metabolism 40:1016-1019; Soulis-Liparota et al., 1991, Diabetes 40:1328-1334; and Edelstein et al., 1992, Diabetologia 35:96-97).
To date, no one has studied or identified 3DG in skin, therefore, the role of 3DG in skin diseases, disorders, or conditions has not been elucidated. Skin aging, wrinkling, and the like, are the subject of much research and there is a long felt need in the art for the development of new methods to treat wrinkling or aging skin, as well as diseased skin. The present invention satisfies this need.